Apicomplexan parasites in the genera Babesia and Plasmodium infect mature erythocytes and cause disease in animals and humans. A principal vaccine strategy is to direct immune responses against merozoite surface proteins and block entry into the host erythrocyte. While parasite proteins involved in invasion have been identified, the function of these proteins and the molecular mechanisms of invasion are major gaps in our knowledge. I propose to use Babesia bovis infection of bovine erythrocytes as a model to investigate the function of merozoite surface proteins in invasion. Based on the ability of antibodies against B. bovis merozoite surface antigen-1 (MSA-1) to prevent merozoite attachment to the erythrocyte and inhibit erythrocyte invasion in vitro, MSA-l has been postulated to mediate merozoite attachment to the erythrocyte. In addition, breakthrough isolates in vaccinated cattle express allelic variants of MSA-1, suggesting selection of MSA-1 variants under immune pressure. Therefore, I am interested in better understanding the role of MSA-l in invasion with the goal of defining conserved molecular targets for immunization. The research outlined in this proposal will test the hypothesis that merozoite surface antigen-1 (MSA-1) of Babesia bovis mediates erythrocyte invasion through binding to the erythrocyte surface. The hypothesis will be tested through the following specific aims: Specific aim 1: Determine if B. bovis MSA-1 binds to the erythrocyte surface. Specific aim 2: Determine if inhibition of MSA-l expression disrupts parasite binding to the erythrocyte. Specific aim 3: Determine if disruption of the msa-l gene inhibits parasite binding to and invasion of erythrocytes. The primary purposes of this research are to increase our understanding of the mechanisms of erythrocyte invasion used by babesial parasites, and to develop a system for investigating the function of other babesia1 molecules using genetic manipulation. The information obtained will expand our knowledge of comparative invasion mechanims used by apicomplexan hemoparasites, including Babesia and Plasmodium spp that infect humans. The results of this project, as well as the transfection techniques that will be developed, will allow us to begin targeting regions of functional significance in these proteins for further study.